Non-spreading pestivirus

ABSTRACT

The invention relates to vaccines used in the eradication or control of pestivirus infections, particularly used in pigs or ruminants. The invention provides nucleic acid, pestivirus-like particles and a pestivirus vaccine, comprising the nucleic acid or particles, which is capable of eliciting a proper immune response without having the ability to spread throughout the vaccinated animal, thereby avoiding the negative consequences of viral spread. Preferably, the immunological response allows for serological discrimination between vaccinated animals and wild-type pestivirus infected animals.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of co-pending International PatentApplication No. PCT/NL00/00153 filed on Mar. 8, 2000 designating theUnited States of America (International Publication No. WO 00/53766published on Sep. 14, 2000), the contents of the entirety of which areincorporated by this reference.

TECHNICAL FIELD

The invention relates to vaccines used in the eradication or control ofpestivirus infections, particularly those used in pigs or ruminants.

BACKGROUND

The genus Pestivirus of the family Flaviviridae conventionally consistsof classical swine fever virus (“CSFV”), Border disease virus (“BDV”),and bovine viral diarrhea virus (“BVDV”). Genomes of several BVDV, BDVand CSFV strains have been sequenced, individual pestiviral proteinshave been expressed and viruses derived from (full-length) DNA copies ofthe RNA genome of BVDV and CSFV have been generated (Renard et al.,1987, EP application 0208672; Collett et al., 1988, Virology 165,191-199; Mendez et al., J. Virol. 72:4737-4745, 1988; Deng and Brock,1992, Virology 1991, 865-679; Meyers et al., 1989, Virology 171,555-567; Moormann et al., 1990, Virology 177, 184-188; Meyers et al.,1989, EP 89104921; Moormann and Wensvoort, 1989, PCT/NL90/00092;Moormann and Van Rijn; 1994, PCT/NL95/00214; Ridpath et al., 1997, VirusRes. 50: 237-243; Becker et al., 1998, J. Virol. 72:5165-5173, Meyers etal., J. Virol. 70:1588-1595, 1996).

The pestivirus genome is a positive-stranded RNA molecule of about 12.5kilobases containing one large open reading frame (“ORF”). The ORF istranslated into a hypothetical polyprotein of approximately 4,000 aminoacids, which is processed by virus- and cell-encoded proteases. The ORFis flanked by two conserved nontranslated regions, which are probablyinvolved in the replication of the genome. The 5′-noncoding region alsoplays a role in initiation of translation.

The polyprotein which is co- and post-translationally processed bycellular and viral proteases contains all the viral structural andnonstructural proteins (for review, see, C. M. Rice: In Fields Virology,Third Edition, 1996 Flaviviridae: The Viruses and their Replication:Chapter 30: pp. 931-959). The viral structural proteins, the capsidprotein C and the envelope proteins E^(ms), E1 and E2, are located inthe N-terminal part of the polyprotein. The nonstructural proteins,including the serine protease NS3 and RNA replicase complex NS5A andNS5B, are located in the C-terminal part of the polyprotein.

Pestiviruses are structurally and antigenically closely related. Todate, pestiviruses such as BDV, BVDV and CSFV have been isolated fromdifferent species, most notably from ruminants and pigs, but infectionof humans has also been reported. All pestiviruses have in common theability to induce congenital infections of fetuses when a pregnantanimal is infected. Such fetal infections occur via transplacentalinfection if the dam undergoes an acute infection during pregnancy or ispersistently infected with a pestivirus (Oirschot, J. T. van, Vet.Microbiol. 4:117-132, 1979; Baker J. C., JAVMA 190:1449-1458, 1987;Nettleton P. F. et al., Comp. Immun. Microbiol. Infect. Dis. 15:179-188,1992; Wensvoort G. and Terpstra C., Res. Vet. Sci. 45:143-148, 1988).

Currently, modified-live, killed and subunit pestivirus vaccines areavailable. Live-virus vaccines have the advantage over the other typesof vaccines of achieving higher levels of immunity without the need ofbooster vaccination. However, disadvantages include the ability ofvaccinal strains to cross the placenta and induce all known consequencesof fetal pestivirus infection (Liess B. et al., ZentralbladVeterinarmed. [B] 31:669-681, 1984). Furthermore, modified-livepestivirus vaccines have been reported to cause immunosuppressiveeffects, probably due to their ability to spread through the vaccinatedanimal and replicate for several days in lymphocytes and neutrophils,thereby causing leukopenia and horizontal spread (Roth J. A. andKaeberle M. L., Am. J. Vet. Res. 44:2366-2372, 1983). Furthermore,epizootics of mucosal disease (a consequence of a persistent BVDVinfection) and of acute BVDV infections have been reported aftervaccination with live-virus vaccines (Lambert G., JAVMA 163:874-876,1973).

Thus, despite the fact that live vaccines are generally considered ashaving the best immunological properties, there are distinct downsidesto using a live pestiviral vaccine in the control and eradication ofpestivirus infections.

These downsides are related to the fact that a conventional livepestiviral vaccine, after inoculation of the animal with the vaccine,undergoes several rounds of replication and spreads through thevaccinated animal. For one thing, this may result in the above-reportedshedding of the virus (horizontal spread), which, after all, is a normalresult of any viral infection, whereby an animal is infected with avirus after which the virus replicates, spreads through the body, mayreplicate again, and eventually is shed from the infected animal tospread to and infect a second, contact, animal.

Even more serious, however, are congenital infections with pestiviruses,causing the so-called vertical spread. Fetuses get infected when thevirus spreads through the body of a pregnant animal and the viruscrosses the transplacental barrier. Depending on the time of gestationand the virulence of the infecting virus, several effects can benoticed. Severe effects include the death of embryos or fetuses,malformations, mummification, stillbirth or perinatal death (Liess B.,Vol. 2 Disease Monographs (E. P. J. Gibss, Editor) Academic Press,London. pp 627-650, 1982). Less virulent virus infections, or infectionslater in gestation, generally result in the birth of congenitallyinfected offspring (van Oirschot J. T. in: Classical swine fever andrelated viral infections, B. Liess (ed) Martinus Nijhoff PublishingBoston pp 1-25, 1988), i.e., calves, lambs, or piglets that arecommonly, persistently infected for life often do not thrive well, areprone to immunosuppression and (sub)clinical disease (such as mucosaldisease with BVDV (Brownlie, J. Arch. Virol. [Suppl. 3]:73-96, 1991))and, last but not least, are a continuing source of infection for therest of the population.

DISCLOSURE OF THE INVENTION

The invention provides nucleic acid, pestivirus-like particles and amodified-live pestivirus vaccine comprising the nucleic acid orparticle(s) which is capable of eliciting a proper immune responsewithout having the ability to spread throughout the vaccinated animal,thereby avoiding the negative consequences of viral spread. Preferably,the immune response allows for serological discrimination betweenvaccinated animals and wild-type pestivirus infected animals.

Viral spread or spread of viral nucleic acid in an inoculated animalcould, in theory, also be prevented by inoculating an animal with apestiviral defective interfering particle (DI) as, for example, knownfrom Meyers et al. (J. Virol. 70:1588-1595, 1996) or Kupfermann et al.(J. Virol. 70:8175-8181, 1996) if one could obtain the DI particles freefrom the helper pestivirus required for their replication, which is, forall practical purposes, near impossible. Inoculating the animal with aDI preparation containing the helper virus as well would defeat all thepurposes; the helper virus would spread throughout the animal, therebysubjecting it to the undesired pestivirus infection, allowing horizontaland vertical transmission. Serological discrimination is thus also notpossible, since antibodies directed against the helper virus would bedetected.

However, even if one succeeded in obtaining the DI particles free of thehelper virus, it still would amount to nothing; the pestiviral DIparticles contain no nucleic acid that allow it to elicit a properimmune response, since the DI nucleic acid essentially does not containthe nucleic acid encoding for structural proteins or immunodominantparts thereof that are responsible for the proper immune response.

In a first embodiment, the invention provides a recombinant nucleic acidderived from a pestivirus from which nucleic acid, a fragment encodingat least one pestiviral protein or substantial part thereof related toviral spread is functionally deleted, the nucleic acid allowing for RNAreplication in a suitable cell and encoding at least one functionalstructural protein or at least one immunodominant part thereof.

“Functionally deleted” herein comprises any insertion, modification ordeletion of the viral genome that results in the production (viatranscription and translation of the nucleic acid in a cell, preferablya cell suitable for the transcription and translation of the nucleicacid, preferably a cell in an animal to be vaccinated) of an at leastfunctionally inactivated viral protein or fragment thereof that in itswild-type state is involved in viral spread, or at least in transmissionto, or viral infection of, cells. Because of the inactivated protein,even when incorporated into the viral particle comprising the nucleicacid, the functional deletion has disabled the particle to enter orinfect a cell which normally, had that protein or functional fragmentbeen functioning properly in the particle, would be infected by theparticle. In this way, although the particle may yet still be formed,the particle is no longer infectious for other cells and can, thus, nolonger contribute via the route of infection to the spread ortransmission of the particle to another cell, notwithstanding the factthat a cell, once infected, may fuse and/or divide, thereby generatingmultiple cells comprising the particle.

The nucleic acid provided by the invention allows for RNA replication ina suitable cell and encodes at least one functional protective proteinor at least one immunodominant part thereof. In a preferred embodimentof the invention, the protective protein is a structural protein, ingeneral, and the immunodominant parts of structural envelope proteinsmount the best immune response of the pestiviral proteins. However, somenon-structural proteins, such as NS3, are also capable of mounting asufficient immune response for some purposes and can, therefore, also beincluded. Thus, although spread-through infection has now beenprevented, the fact that RNA replication is possible allows for one ormore rounds of transcription and translation in the cell ofimmunologically dominant proteins against which a vaccinated animalmounts an immune response through which it is at least partly protectedagainst the consequences of infection with a wild-type pestivirus. Thetranslated protein(s) or fragment(s) thereof in themselves (is) areresponsible for the immune response and may also become part of avirus-like particle, even comprising the replicate RNA, but the particleis not infectious due to the fact that one essential functional featureof the functionally deleted protein is missing.

Although in one embodiment the nucleic acid as provided by the inventionmay comprise DNA, as to allow for DNA vaccination, in anotherembodiment, the invention provides nucleic acid wherein the nucleic acidis RNA to allow for RNA vaccination. Such RNA is, for example, packagedinto a virus-like particle in a complementing cell, as provided by theinvention, provided with a functional protein or fragment (derived fromthe complementing cell) responsible for virus-cell interactions allowingthe particle to enter or infect a suitable cell, or may be introducedinto the animal's cells otherwise, such as via the intradermal route.

In a preferred embodiment, the invention provides a nucleic acid whereinthe functional deletion is in a fragment encoding an envelope protein.Essential to infection with pestiviruses is the interaction of viralstructural proteins with the surface or a receptor of the susceptiblecell. It is through this interaction that the infection takes place.Especially envelope proteins E2 and/or E^(ms) provide for thisinteraction, and functionally deleting at least one of these envelopeproteins or functional fragments thereof (in particular those fragmentsinvolved in receptor or surface interaction) leads to obstruction ofinfectivity.

Several examples of such functional deletions in a nucleic acid-encodingprotein related to viral spread are given in the detailed description ofthe invention. An example comprises a modification of acysteine-encoding nucleic acid codon, whereby a conformational change isinduced in a fragment of a pestiviral protein, preferably an envelopeprotein, in such a way that the functionally deleted protein, whenincorporated in the particle, has disabled the particle to enter anotherwise accessible cell. One example is the modification of a codonresulting in a cysteine change, for example, at amino acid position 422,or, for that matter, at position 381, of the amino acid sequence of theE^(ms) protein of CSFV, or at functionally corresponding locations inthe E^(ms) protein of CSFV or other pestiviruses, which, for example,obtained by sequence comparison, are also provided by the invention.Another example comprises deleting larger fragments of a nucleic acidencoding a pestiviral protein, for example, by deleting at least afragment encoding approximately corresponding positions 170-268 or otherfunctionally related fragments of the capsid proteins C of CSFV or otherpestiviruses or by deleting at least a fragment encoding approximatelycorresponding positions 500-665 or other functionally related fragmentsof the E1 proteins of CSFV or other pestiviruses, or comprises deletingfragments encoding, etc. Another example comprises deleting largerfragments of a nucleic acid encoding a pestiviral protein, for example,by deleting at least a fragment encoding corresponding positions 381,422, 381-422, 405-436, 422-436, 422-488 or 273-488 or other functionallyrelated fragments of the E^(ms) protein of CSFV or other pestiviruses,or comprises deleting fragments encoding corresponding positions698-1008 or 689-1062 in the E2 protein of CSFV or other functionallyrelated fragments of the E2 protein of CSFV or other pestiviruses.

In a much preferred embodiment, the invention provides a nucleic acidwherein the functional deletion comprises an immunodominant part of theprotein. For example, deleting a fragment corresponding to about aminoacid positions 422-436 or 422-488 of the E^(ms) protein, orcorresponding to about amino acid positions 693-746, 785-870, 689-870 or800-864 of the E2 protein or any other fragment related to a discernibleimmune response against the protein has the additional advantage that adiscernible vaccine is provided.

By deleting the serologically discernible fragment, in the end, a markervaccine is obtained that allows for serological discrimination betweenvaccinated animals and animals infected with a wild-type pestivirus.

In constructing a vaccine, one has to take into account what (type of)serological test is preferred once the vaccine is employed in the field.For CSFV, it preferably should be genotype specific, which blocks usingdiagnostic tests based on NS3. However, selecting E2 or E^(ms) asdiagnostic antigens hampers developing a vaccine which uses theprotective properties of these proteins. The invention surprisinglyprovides a pestivirus vaccine in which a protein, preferably an envelopeprotein comprising a specific immunodominant part, in general, thoughtresponsible for generating protection, has been (functionally) deleted,allowing serological discrimination surprisingly without seriouslyhampering protective properties.

The protective properties are optimally provided by a nucleic acidaccording to the invention having a fragment encoding a protectiveprotein that is a functional structural protein, more preferably afunctional envelope protein or at least one immunodominant part thereof.Most preferred by the invention is a nucleic acid comprising a fragmentencoding a functional deletion in one pestiviral envelope protein, forexample, E2 or E^(ms), respectively, and further comprising a nucleicacid encoding another protective envelope protein, or immunodominantpart thereof, for example, E^(ms) or E2 or part thereof, respectively.

In a further embodiment, the invention provides a nucleic acidadditionally comprising a non-pestivirus nucleic acid fragment, therebyproviding a nucleic acid encoding heterologous protein or fragmentsthereof. Heterologous protein (fragments) may be used as a marker or maybe used to elicit a (protective) immune response. Marker sequences arepreferably highly antigenic and, in one embodiment of the invention,preferably derived from a (micro)organism not replicating in animals.They may encode known complete gene products (e.g., capsid or envelopeproteins or antigenic parts of gene products (e.g., epitopes). Markersequences may also encode artificial antigens not normally encounteredin nature, or histochemical markers like Escherichia coliβ-galactosidase, Drosophila alcohol dehydrogenase, human placentalalkaline phosphatase, firefly luciferase and chloramphenicolacetyltransferase. Also provided is a nucleic acid wherein thenon-pestivirus fragment is derived from a pathogen encoding one or moreprotein (fragments) inducing protective immunity against disease causedby the pathogen, such as a fragment derived from parvovirus,coronavirus, porcine respiratory and reproductive syndrome virus,herpesvirus, influenza virus, and numerous other pathogens known in theart. Also provided is a nucleic acid wherein the non-pestivirus fragmentis derived from a cytokine inducing immuno-regulating or -stimulatingsignals when expressed. Numerous cytokines are known in the art, such asinterleukines, interferons and tumour necrosis or colony-stimulatingfactors.

The invention further provides a nucleic acid according to the inventionwherein the suitable cell comprises a nucleic acid construct encoding atleast the pestiviral protein or substantial part thereof related toviral spread. Such a suitable cell, which is also provided by theinvention, comprises a cell comprising a recombinant nucleic acidencoding at least one pestiviral protein or substantial part thereofrelated to viral spread and allows packaging the pestiviral protein orsubstantial part thereof in a pestivirus-like particle. Such a packagingor complementing cell, according to the invention, allows nucleic acidor replicate nucleic acid according to the invention to be part of apestivirus-like particle, wherein a substantial part of the protein(fragments) composing the particle is derived from translation andtranscription in the cell of nucleic acid according to the invention,being complemented with a protein (fragment) related to viral spreadderived from the nucleic acid construct that is also expressed in thecomplementing cell. Such a protein (fragment) can be transientlyexpressed from a nucleic acid construct or can be expressed from ahelper virus, but preferred is a cell according to the invention whereinthe pestiviral protein or substantial part thereof related to viralspread is stably, either inducably or constitutively, expressed from,for example, a self-replicating nucleic acid or from the cellular genomeintegrated nucleic acid.

The invention also provides a method for obtaining a pestivirus-likeparticle comprising transfecting such a cell according to the inventionwith a nucleic acid according to the invention, further comprisingallowing the nucleic acid to replicate in the cell, further comprisingallowing replicated nucleic acid to be part of a particle comprising atleast the pestiviral protein or part thereof derived from the cell, andfurther comprising harvesting the particle. Such a use of a nucleic acidaccording to the invention, or a cell according to the invention, inproducing a pestivirus-like particle is provided by the invention. Bytransfecting the cell with nucleic acid according to the invention andallowing the nucleic acid to replicate, a replicated RNA of the nucleicacid is packaged in the pestivirus-like particle, the particle alsocomprising a functional protein or set of proteins related to viralspread, at least partly derived from the nucleic acid construct withwhich the complementing or packaging cell has also been provided.Likewise, the invention provides a pestivirus-like particle obtainableby a method according to the invention; for example, the inventionprovides a pestivirus-like particle (or a multitude of such particles)comprising nucleic acid derived from a pestivirus, from which nucleicacid, a fragment encoding at least one pestiviral protein, orsubstantial part thereof related to viral spread, is functionallydeleted, the nucleic acid allowing for RNA replication in a suitablecell and encoding at least one functional structural protein or at leastone immunodominant part of an immunodominant structural protein. Theparticle may be derived from one type of pestivirus but can also be aso-called hybrid particle, wherein its genome and part of itsconstituting protein (fragments) is (are) derived from one typepestivirus, such as BVDV or BDV, but wherein complementing protein(fragments) are derived from another type pestivirus, such as CSFV. Theparticle, having been produced in the packaging or complementing cell asprovided, is itself infectious and, thus, capable of entering a suitablesecond cell, such as a non-complementing cell capable of being infectedwith a pestivirus type in general, or such as a susceptible cell in ananimal to be vaccinated. When replicating in the second cell (which isnon-complementing) a new particle is produced that, however, lacks thepossibilities to infect yet another cell and is, thus, unable to spreadby infection. Thus, when the particles produced in a complementing cellare used to infect an animal, such as when used in or as a vaccine, theparticles will infect suitable cells in the vaccinated animal, fromwhich, however, no new particles that spread by infection to other cellsare generated, thereby demonstrating the requirements of a non-spreading(non-transmissible) vaccine.

The invention also provides a method for obtaining a non-spreadingpestivirus vaccine comprising obtaining a multitude of particles by amethod according to the invention and preparing a suspension of theparticles in a suitable diluent. Suitable diluents are known in the artand preferably on a watery basis, such as a (buffered) salt solution or(growth) medium. The invention also provides a method for obtaining anon-spreading pestivirus vaccine comprising obtaining a multitude ofparticles by a method according to the invention and preparing asuspension of the particles in a method comprising combining thesuspension with an adjuvant. Suitable adjuvants are water-oil emulsions,aluminum salts or other adjuvants known in the art, see, for example,Vogel F. R. and Powell M. F, A compendium of adjuvants and excipients.In: Vaccine design. (eds) Powell and Newmann, PharmaceuticalBiotechnology Series, Plenum, N.Y., 1994. The invention thus provides anon-spreading pestivirus vaccine obtainable by a method according to theinvention. The invention provides such a vaccine comprising a nucleicacid according to the invention or a pestivirus-like particle accordingto the invention, for example, further comprising an adjuvant. Optimalefficacy of the vaccine is achieved if its nucleic acid is targeted tosuitable antigen presenting cells. Replication and translation of theviral RNA in these cells will result in processing of viral antigen foroptimal presentation to the immune system.

In one embodiment of the invention, the vaccine consists ofpestivirus-like particles produced in a complementing packaging cellwhich, with or without an adjuvant, are applied to the animal viadifferent routes such as, but not limited to, intranasal, intramuscular,intradermal or intravenous vaccination, or a combination of routes. Inanother embodiment of the invention, the vaccine consists of essentiallynaked DNA or RNA according to the invention which, with or without anadjuvant, is preferably applied to the animal via the intradermal route.However, alternative routes such as, but not limited to, theintramuscular route are suitable.

Such a vaccine is provided wherein the nucleic acid is derived from anypestivirus (vaccine) strain from which (full-length) cDNA and infectiouscopies thereof are, or can be, provided, such as C-strain virus or fromanother pestivirus such as another vaccine-type or wild-type of aclassical swine fever virus, a bovine viral diarrhea virus or a Borderdisease virus, or chimeric virus. For simplicity's sake, the numberingof the C-strain sequence is used herein for all pestivirus sequences. Infact, in other pestivirus sequences, the numbering of the E^(ms) and theE2 proteins in the (poly)protein may differ slightly due to lengthdifferences in the (poly)protein sequences of pestivirus strains. Basedon homology, the N and C termini of the E2 or E^(ms) sequence of anypestivirus strain can, however, easily be determined, such as shown byRumenapf T. et al., J. Virol, 67:3288-3294, 1993, or Elbers K. et al.,J. Virol. 70:4131-4135, 1996.

The invention also provides a method for controlling and/or eradicatinga pestivirus infection comprising vaccinating at least one animal with avaccine according to the invention. The vaccination serves to prevent ormitigate a wild-type pestivirus infection which the animal may have beenor may be confronted with due to the presence of wild-type virus in itssurroundings. Since no spread or shedding of the vaccine occurs, thevaccinated animal can be safely vaccinated, even when pregnant; no riskof congenital vaccinal infections of its fetus, or shedding of thevaccine from the vaccinated to a non-vaccinated animal, is present dueto the non-spreading nature of the vaccine.

Additionally, the invention provides a method for controlling and/oreradicating a pestivirus infection comprising testing an animalvaccinated with a vaccine according to the invention for the presence ofantibodies specific for a wild-type pestivirus. In a preferredembodiment, the method for controlling such a vaccine is used as amarker vaccine. The use of such a marker vaccine as provided by theinvention allows serological discrimination between vaccinated andfield-virus infected animals, and thereby a controlled elimination ofthe virus. For serological discrimination of pestivirus genotypes, itdoes not suffice to provide protection with a vaccine comprising theprotective proteins E2 or E^(ms) but not the NS3 protein and detectinginfections with tests based on NS3. NS3 is not genotype-specific; atleast it does not elicit genotype-specific antibodies to allowdiscrimination between genotypes. Although no objection can be seen atusing the tests to diagnose BVDV or BDV infections, such diagnostictests can, thus, hardly be used in the aftermath of vaccinationcampaigns against, for example, CSFV in pigs. Circulating NS3-BDV orNS3-BVDV antibodies will cause a plethora of false-positive results,leading to suspicions of CSFV infections when, in fact, there aren't anyin the pig population tested. Preferably, tests are used to detectantibodies against E2, or serologically discernible fragments thereof,when the functional deletion is in the nucleic acid fragment encodingthe E2 protein and the protective protein mainly comprises E^(ms)protective protein or fragments thereof, optionally supplemented withother protective protein (fragments), or vice versa; tests are used todetect antibodies against E^(ms), or serologically discernible fragmentsthereof, when the functional deletion is in the nucleic acid fragmentencoding the E^(ms) protein and the protective protein mainly comprisesE2 protein or fragments thereof, optionally supplemented with otherprotective protein (fragments).

The invention also provides an animal vaccinated with a non-spreading(non-transmissible) pestivirus vaccine according to the invention. Sincesuch an animal bears no risk of spreading the vaccine to contactanimals, or to its fetus(es), such an animal has considerable advantagesover animals vaccinated with conventional pestivirus vaccines. It can,for example, be traded during that period shortly after vaccinationwhere trade otherwise has to be restricted due to the risk of shedding.

The invention is further explained in detail below without limiting theinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 Schematic representation of E^(ms) of CSFV strain C (top) andoverview of E^(ms) plasmids (bottom). Domains of RNase activity areshown by closed bars. Positions of cysteines are indicated with blackdots.

-   ^(a) Positions of the deletions or point mutations with respect to    the amino acid sequence of the open reading frame (ORF) of CSFV    strain C (Moormann et al., J. Virol. 1996, 70: 763-770).-   ^(b) Cysteine-to-serine mutations are depicted with white dots.-   ^(c) The recombinant E^(ms) in these plasmids do not harbor a    C-terminal HA tag.-   NA: not available.

FIG. 2 Schematic representation of the construction of the full-lengthDNA copies pPRKflc23 (A) and pPRKflc22 (B) harbouring E^(ms) deletions:The amino acid sequence numbering is of the open reading frame (ORF) ofthe CSFV strain C (Moormann et al., 1996, J. Virol., 70: 763-770). PCRprimers are indicated with solid lines and designated: p(number);N^(pro), autoprotease; C, core protein; E^(ms), E1 and E2 envelopeproteins; 5′, 5′ non-coding region; 3′, 3′ non-coding region, Amp,ampicillin resistance gene; CIP, calf intestinal phosphatase; Kan,kanamycin resistance gene; ORF: open reading frame; PNK, polynucleotidekinase; PhCMV, promoter-enhancer sequence of the immediate early gene ofhuman cytomegalovirus; T7, bacteriophage T7 promotor. pPRKflc2 is thewild-type full-length cDNA copy of the CSFV strain C. (B) PlasmidpPRKc129 was the template for the first PCR of E^(ms). The Nar I site ofthis PCR product and the Cla I site of the hemagglutinin (HA) epitopehave compatible ends. These two PCR fragments were inserted into the BglII/Sal I digested vector pPRKc16 via a three-point ligation. See textfor detailed information on the construction of the full-length DNAcopies and the primer sequences.

FIG. 3 Characterization of recombinant viruses.

-   ^(a) Positions of cysteines are indicated with black dots,    cysteine-to-serine mutations are depicted with white dots.-   ^(b) Positions of the deletions or mutations with respect to the    amino acid sequence of CSFV strain C (Moormann et al., J. Virol.    1996, 70: 763-770).-   ^(c) Supernatants from SK6c26-infected cells were used for infection    of SK6 and SK6c26 cells. The cells were immunostained with    E^(ms)-specific antibodies (R716 and C5) or an E2-specific Mab (b3)    and were scored as positive (+) or negative (−).-   ^(d) Viruses are considered to be infectious viruses if supernatant    of the infected cells can infect SK6 or SK6c26 cells. Spread of    virus via cell-to-cell spread and spread of virus due to division of    cells is not considered as infectious virus.

FIG. 4 RT-PCR of SK6c26 cells infected with Flc22, Flc23 and Flc2 withprimers flanking E^(ms) and E2. (−) negative control: mock infectedSK6c26 cells; M: 200 bp marker.

FIG. 5 Growth kinetics of the recombinant CSFV viruses Flc22, Flc23 andthe wild-type virus Flc2. Subconfluent monolayers of SK6c26 cells wereinfected at a multiplicity of 0.1. Viruses were adsorbed for 1.5 hr.Virus titers of the cell lysates and supernatant at various time pointswere determined by end point dilution on SK6c26 cells.

FIG. 6 E^(ms) amino acid sequence of the recombinant Flc22, Flc23 andthe wild-type strain Flc2, |: indicates position of deletion.

FIG. 7 Schematic representation of pPRKflc23 harbouring the HA epitope,a non-pestivirus sequence. The HA epitope is flanked by the 5 utmostN-terminal amino acids and 6 utmost C-terminal amino acids of E^(ms).

FIG. 8 Schematic representation of the construction of the full-lengthDNA copies pPRKflc4 (A) and pPRKflc47 (B). The amino acid sequencenumbering is of the open reading frame (ORF) of the CSFV strain C(Moormann et al., 1996, J. Virol.). PCR primers are indicated with solidlines and designated: p(number); N^(pro), autoprotease; C, core protein;E^(ms), E1 and E2 envelope proteins; p7: p7 protein, NS, nonstructuralprotein, 5′, 5′ non-coding region; 3′, 3′ non-coding region, Amp,ampicillin resistance; Kan, kanamycin resistance. pPRKflc2 is thewild-type full-length DNA copy of the CSFV strain C. (A) The E2 gene ofplasmid pPAB16 was inserted in plasmid pPRKc129 by NgoMI/BglIIdigestion. (B) pPRKflc2 was the template for PCR amplification withprimers p1195 and p403. See text for detailed information on theconstruction of the full-length DNA copies and the primer sequences.

EXAMPLES Example 1

Construction and Characterization of Recombinant CSFV Strains Flc22,Flc23, Flc30, Flc31, Flc32, and Flc33

Materials and Methods

Cells & Viruses

Swine kidney cells (SK6-M, EP 0 351 901 B1) were grown in Eagle's basalmedium containing 5% fetal bovine serum, glutamine (0.3 mg/ml), and theantibiotics penicillin (200 U/ml), streptomycin (0.2 mg/ml), andmycostatin (100 U/ml). Fetal bovine serum was tested for the absence ofBVDV and BDV antibodies as described previously (Moormann et al., 1990,Virology 177:184-198).

Recombinant CSFV strain C viruses Flc22, Flc23, Flc30, Flc31, Flc32, andFlc33 were grown and prepared as described earlier (Moormann et al.,1996, J. Virol. 70: 763-770) with a slight modification, the growthmedium of SK6 cells was changed in supplemented Eagle's basal medium.Virus stocks were prepared by passaging the virus eight to ten times onSK6c26 cells. The obtained virus titres ranged from 5.0 to 5.8TCID50/ml.

Construction of a Stable SK6 Cell Line Expressing E^(ms)

Plasmid pPRKc16 contains the E2 gene of CSFV strain C under control ofthe transcription and translation signals of expression vectorpEVhisD12. Plasmid pEVhisD12 is a vector that contains promoter/enhancersequences of the immediate early gene of the human cytomegalovirusfollowed by a translation initiation codon and the histidinoldehydrogenase gene (hisD) under control of the SV40 early promoter,which can be used as a selective marker (Peeters et al., 1992, J. Virol.66: 894-905). The E^(ms) gene of the CSFV strain C was amplified by PCRreaction with primers p974 5′ AAG AAA AGA TCT AAA GCC CTA TTG GCA TGG 3′(SEQ ID NO:1) and p976 5′ TT GTT ACA GCT GCA TAT GTA CCC TAT TIT GCT TG3′ (SEQ IC NO:2). After BglII digestion, the PCR fragment was ligatedinto the vector pPRKc16, which was digested with SalI, filled in, andsubsequently digested with BglII. The resulting plasmid pPRKc26 containsthe E^(ms) gene of the CSFV strain C.

For transfection of SK6 cells with pPRKc26, lipofectamine (20 μg)(Gibco-BRL) was diluted in 50 μl of Optimem-I (Gibco-BRL) mixed withplasmid DNA (1 μg) diluted in 50 μl Optimem-I (Gibco-BRL) and thismixture was allowed to settle for 15 min at room temperature. SK6 cellsgrown in 10 cm² tissue culture plates were washed with Optimem-I. FreshOptimem-I was added (0.5 ml), followed by the DNA transfection mixture.After 4 h of incubation at 37° C., the transfection mixture was removedand the wells were supplied with medium containing 5 mM histidinol.After 24 h of incubation at 37° C., cells were trypsinized and plated ona 90 mm2 plate. Medium was replaced every 34 days. After 15 days, singlecolonies were picked and plated into 2 cm² plates. Expression of E^(ms)was determined by immunostaining of the cells with Mabs C5 (Wensvoort1989, Thesis, University of Utrecht) directed against E^(ms) of CSFVstrain C. A second round of cloning was performed by trypsinizing andplating the cells in ten-fold dilution in microtiter plates in mediumcontaining 5 mM histidinol. Wells with individual colonies weretrypsinized and expression of E^(ms) was determined by immunostaining(Wensvoort et al., 1988, Vet. Microbiol. 17, 129-140) the cells with MabC5. The established SK6 cell line constitutively expressing E^(ms) wasnamed SK6c26.

Characterization of the Stable Cell Line SK6c26

E^(ms) expression of the cell line SK6c26 line was tested in animmunoperoxidase staining with E^(ms)-specific monoclonal antibodies(Mabs) C5, specific for E^(ms) of strain C (Wensvoort 1989, Thesis,University of Utrecht), 140.1 and 137.5 directed against E^(ms) of CSFVstrains C and Brescia (de Smit et al., unpublished data), and apolyclonal rabbit serum, R716 (Hulst et al., J. Virol. 1998, 72:151-157). The RNase activity of the E^(ms) expressed in the SK6c26 cellline was measured by a modification of the method of Brown and Ho (PlantPhysiol. 1986, 82: 801-806) as described by Hulst et al., (J. Virol.1998, 72: 151-157). The amount of E^(ms) was determined by an indirectELISA based on Mab C5 as coating antibody and horseradish peroxidaseconjugated Mab 140.1 as detection antibody as described by Hulst et al.(J. Virol. 1998, 72: 151-157).

Construction of Recombinant CSFV E^(ms)

-   -   pPRKc5 (Hulst et al., Virol. 1998, 72: 151-157) is a pEVhisD12        derivative which contains the nucleotide sequence of the        autoprotease and structural genes of CSFV strain C, without        E^(ms) (Npro-C and E1-E2, amino acids (a.a.) 5-267 and 495-1063        of the amino acid sequence of CSFV strain C) (Moormann et al.,        1996, J. Virol. 70: 763-770). A unique Stu I site was introduced        in pPRKc5 at the position where E^(ms) was deleted.

Two complementary oligomers, the forward oligomer p1135 (5′CCG AAA ATATAA CTC AAT GGT TTG GCG CTT ATG 3′(SEQ ID NO:3)) and the reverseoligomer p1136 (5′CAT AAG CGC CAA ACC ATT GAG TTA TAT TTT CGG 3′ (SEQ IDNO:4)) were phosphorylated with T4 DNA kinase, hybridized and insertedvia ligation in an alkaline phosphatase-treated StuI-digested vectorpPRKc5. This construct was named pPRKc48. This construct harbors thefive utmost N-terminal amino acids and the six utmost C-terminal aminoacids of E^(ms) (deletion a.a 273-488) (FIG. 1 and FIG. 2A).

A deletion of amino acids 422 to 488 in E^(ms) of strain C wasaccomplished by PCR amplification of the E^(ms) gene using the forwardprimer p974 and reverse primer p1120 5′ GAC GGA TTC GGC ATA GGC GCC AAACCA TGG GCT CTC TAT AAC TGT AAC 3′ (SEQ ID NO:5). The HA epitope, aminoacid sequence YPYDVPDYA (SEQ ID NO:6) (Wilson et al., Cell 1984, 37,767-778), was constructed by annealing the 3′ complementary nucleotidesof p1124 '5 GAC AGA TCT ATC GAT TAC CCA TAC GAT GTT CCA GAT 3′ (SEQ IDNO:7) and p1255 5′GAC GTC GAC GGA TCC AGC GTA ATC TGG AAC ATC 3′ (SEQ IDNO:8) (underlined is the HA sequence) and filling in the 5′ singlestrand nucleotides in a PCR with Vent polymerase (New England Biolabs).The HA epitope PCR product was digested with ClaI/SalI, and the E^(ms)PCR product was digested with BglII/NarI. The two digested PCR productswere ligated via a three-point ligation into the vector pPRKc16 whichwas digested with BglII and SalI. This resulted in plasmid pPRKc43containing a recombinant E^(ms) with a deletion of amino acids 422 to488 with C terminally an HA epitope (FIG. 1 and FIG. 2B). After PCRamplification of plasmid pPRKc43 with the forward primer p935 (5′CCG AAAATA TAA CTC AAT GG 3′ (SEQ ID NO:9)) and the reverse primer p925 (5′CATAAG CGC CAA ACC AGG TT 3′ (SEQ ID NO:10)), the PCR product wasphosphorylated with T4 DNA kinase and subsequently ligated into thealkaline phosphatase treated StuI digested vector pPRKc5. The resultingconstruct harbouring the nucleotide sequence of the autoprotease, thestructural proteins of strain C and the recombinant E^(ms) lacking aminoacids 422 to 488 was named pPRKc50 (FIG. 2B).

A deletion mutant lacking amino acids 436 to 488 of E^(ms) of strain Cwas accomplished by PCR amplification of the E^(ms) gene using theforward primer p974 and reverse primer p1121 (5′ GAC GGA TTC GGC ATA GGCGCC AAA CCA ATC CCC ATA CAA GGT ATC CTC 3′ (SEQ ID NO:11)). AfterBglII/NarI digestion, the fragment was ligated with the ClaI/SalIdigested HA epitope PCR product via a three-point ligation in the vectorpPRKc16, which was digested with BglII/SalI. The resulting plasmid,pPRKc42, was PCR amplified with the forward primer p935 and reverseprimer p925. This PCR product was phosphorylated with T4 DNA kinase andsubsequently ligated into the alkaline phosphatase-treated StuI-digestedvector pPRKc5. The resulting construct harbouring the nucleotidesequence of the autoprotease, the structural proteins of strain C andthe recombinant E^(ms) lacking amino acids 436-488 is named pPRKc49.

A deletion mutant lacking amino acids 422 to 436 was made by PCRamplification of plasmid pPRKc129 with the primers p1147 (5′ CAA ACT GCCGCA CTC ATG TGG GCT CTC TAT AAC TGT 3′ (SEQ ID NO:12)) and p925. Then,the obtained PCR product was isolated from agarose gel and used asreverse primer in a second PCR reaction with primer p935 as forwardprimer for amplification of pPRKc129. This second PCR product waskinated and ligated into the alkaline phosphatase-treated StuI-treatedvector pPRKc5, resulting in plasmid pPRKc51.

A cysteine (CYS) to serine (SER) substitution atamino acid position 422was constructed by PCR amplification of pPRKc129 with the forward primerp1140 5′ GAG AGC CCT TCG AAT TTC AAT GT 3′ (SEQ ID NO:13)) and thereverse primer p925. Further steps were equal to those of the deletionmutant lacking amino acids 422 to 436. Plasmid pPRKc52 contains theautoprotease gene and the structural proteins of strain C, with amutated E^(ms) containing a CYS-to-SER substitution at pos 422.

A cysteine to serine mutation at amino acid position 405 was constructedby PCR amplification of pPRKc129 with the forward primer p1148 (5′CCTGAC CGG TTC GAA GAA AGG GAA-3′ (SEQ ID NO:14)) and the reverse primerp925. Further steps were equal to those of the deletion mutant lackingamino acids 422 to 436. Plasmid pPRKc54 contains the autoprotease geneand the structural proteins of strain C, with a mutated E^(ms)containing a CYS-to-SER mutation at pos 405.

A cysteine to serine substitution at amino acid position 381 wasconstructed by PCR amplification of pPRKc129 with the forward primerp1149 (5′ TGC GCT GTG ACT AGT AGG TAC GAT AAA-3′ (SEQ ID NO:15)) and thereverse primer p925. Further steps were equal to those of the deletionmutant lacking amino acids 422 to 436. Plasmid pPRKc56 contains theautoprotease gene and the structural proteins of strain C, with amutated E^(ms) containing a CYS-to-SER mutation at position 381.

Clones in which the mutated E^(ms) gene were inserted in the rightorientation were transfected into SK6 cells and tested for expression ofE^(ms) and E2 by immunostaining with antibodies against E^(ms) (C5,140.1, 137.5, R716) and E2 specific Mabs b3 and b4 (Wensvoort 1989, J.Gen. Virol. 70:2865-2876).

Construction of Full-Length CSFV Constructs Harbouring E^(ms) DeletionMutants

A ClaI/NgoMI fragment of pPRKc48 and pPRKc50 was isolated and ligatedinto the ClaI/NgoMI digested vector pPRKflc2, formerly named pPRKflc133(Moormann et al., 1996, J. Virol. 70: 763-770) and the resultingfull-length cDNA CSFV strain C E^(ms) mutants were named pPRKflc23 andpPRKflc22. The complete construction scheme of the full-lengthconstructs pPRKflc22 and pPRKflc23 is depicted in FIGS. 2A and 2B and anoverview of the E^(ms) plasmids is given in FIG. 1.

Similarly, the 422-436 deletion mutant of pPRKc51 and the CYS-to-SERsubstitutions of pPRKc52, pPRKc54, pPRKc56, were transferred to thevector pPRKflc2 via a ClaI/NgoMI digestion, resulting in the recombinantfull-length cDNA clones pPRKflc30, pPRKflc31, pPRKflc32, and pPRKflc33,respectively.

Isolation of Recombinant Viruses

Plasmid DNA from pPRKflc22, pPRKflc23, pPRKflc30, pPRKflc31 pPRKflc32,and pPRKflc33 was purified on columns (Qiagen) and linearized with XbaI.The DNA was extracted with phenol-chloroform, precipitated with ethanol,and dissolved in water. RNA was transcribed from the linearized plasmid(1 μg) in a 100 μl reaction volume containing 40 mM Tris HCl (pH 7.5), 6mM MgCl2, 2 mM spermidine, 10 mM dithiothreitol, 40 U rRNAsin (Promega),0.5 mM each rNTP, and 35 U T7 RNA polymerase (Pharmacia). After 1 hincubation at 37° C., 10 U RNase-free DNaseI (Pharmacia) was added andthe mixture was incubated for another 15 min. RNA was extracted withphenol-chloroform, precipitated with ethanol, and dissolved in 10 μlwater.

For RNA transfection, 10 μg Lipofectin was diluted in 50 μl ofOptimem-I. After a 45 min incubation at room temperature, 1 μg RNAdiluted in 50 μl Optimem-I was added and incubated for an additional 15min. SK6c26 cells grown in 10 cm² tissue culture plates were washed withOptimem-I and incubated with the RNA transfection mixture for 4 h at 37°C. Then, the wells were supplied with fresh medium and incubated for 4days at 37° C. RNA transfection was performed in duplicate. One samplewas immunostained with Mabs b3/b4 specific for E2. When the E2immunostaining proved to be negative, the duplicate sample was passagedand split into two samples. One of these samples was used forimmunostaining four days after passaging. From wells in which E2expression was observed, supernatant was applied onto fresh SK6c26 orSK6 cells to determine the presence of infectious virus. After fourdays, the monolayers were fixed and immunostained as described above.

Growth Kinetics of Flc22 and Flc23

Growth kinetics of the viruses was determined in SK6c26 cells.Subconfluent monolayers in M24 wells were infected at a multiplicity ofinfection of 0.1. Viruses were adsorbed for 1.5 h. Before cells weresupplied with fresh medium, the first sample at time point zero wascollected. At 0, 1, 2, 3, 4, 5, 6, and 7 days after infection the M24plates were freeze/thawed twice and clarified by centrifugation for 10min at 5000×g at 4° C. Virus titres (log TCID50 per milliliter) of totallysates (cell lysates plus supernatant) were determined on SK6c26 cells.

Characterization of Recombinant E^(ms) Viruses

The E^(ms) genes of Flc22 and Flc23 were sequenced. Therefore,cytoplasmic RNA of SK6c26 cells infected with these respective viruseswas isolated using the RNeasy total RNA isolation kit (Qiagen). DNAfragments covering the E^(ms) genes were analyzed by RT-PCR usingprimers p1154 (5′ GTT ACC AGT TGT TCT GAT GAT 3′ (SEQ ID NO:16)) andp305 (5′ GGG GTG CAG TTG TTG TAT CCA 3′ (SEQ ID NO:17)) amplifyingnucleotide sequences 865 to 1920, analyzed on a 1.5% agarose gel in1×TAE, and purified on Costar Spin-X columns. An RT-PCR of the E2 genewas performed with primer pair p307 (TGG AAT GTT GGC AAA TAT GT (SEQ IDNO:18)) and p304 (CAC TTA CCT AT[A,G] GGG TAG TGT GG (SEQ ID NO:19))amplifying nucleotide positions 2200-3174.

Sequences of the purified PCR fragments were determined by PCR cyclesequencing using the Big dye dRhodamine terminator ready reaction cyclesequencing kit (PE) according to the manufacturer's conditions withflanking primers and analyzed on a 310 ABI PRISM genetic analyzer.

In addition, the recombinant viruses were characterized by animmunoperoxidase monolayer assay. For this, SK6 cells were infected withthe recombinant viruses and Flc2. After incubation for 4 days at 37° C.,monolayers were immunostained with Mabs specific for CSFV E2 (Mabsb3/b4), CSFV E^(ms) Mabs 140.1, C5, 137.5 and a polyclonal rabbit serumagainst E^(ms) R716.

Virus neutralization index (log reduction of virus titre [TCID50/ml] byneutralizing serum) was determined at a 1:250 dilution of serum 716specifically directed against E^(ms) of CSFV strain C and at a 1:1000dilution of a pig serum 539 specifically directed against E2 of CSFVstrain Brescia (Hulst et al., Virol. 1998, 72: 151-157). The virusstocks of Flc2, Flc22 and Flc23 were titrated by endpoint dilution inthe presence or absence of these CSFV neutralizing antibodies.

Results

Transient Expression of Recombinant E^(ms) in SK6 Cells

Previous studies showed that the antibodies raised against CSFV E^(ms)do not inhibit RNase activity (Hulst et al., 1998, J. Virol.72:151-157). The active domains of the E^(ms) RNase are located in theN-terminal half of the protein (Schneider et al., 1993, Science 261:1169-1711, Hulst et al., 1994, Virol. 200: 558-565). (See FIG. 1 for aschematic representation of E^(ms).) Pepscan analysis did not reveal anylinear epitopes on E^(ms) for the antibodies C5, 140.1 and 137.5 (datanot shown), showing that the epitopes are conformational. Therefore, aset of E^(ms) recombinants were constructed with deletions of differentlengths in the C-terminus (FIG. 1). These deletion mutants wereconstructed in an expression plasmid pPRKc5 harbouring the nucleotidesequence of the autoprotease and the structural genes(Npro-capsid-E1-E2) without E^(ms). Expression of these constructsenabled us to use the E2 gene as control for a correct open readingframe since the E2 gene is located C-terminally of the E^(ms) gene. SK6cells were transfected with these plasmids and an immunoperoxidasestaining was performed 24 hours after transfection (Table 1).

Cells transfected with the plasmid pPRKc49, harbouring a deletion fromamino acid positions 436 to 488, can be specifically immunostained withall antibodies recognizing CSFV E^(ms) (Mab C5, 140.1, 137.5) and thepolyclonal serum R716, like the wild-type plasmid pPRK83. PlasmidpPRKc50, however, harbouring a deletion from position 422 to 488, is notrecognized anymore by the antibodies against E^(ms), but is positivewith Mabs b3/b4 against E2. These results show the presence of adistinct antigenic domain on E^(ms) and/or an important role for aminoacids 422 to 436 for either binding or conformation of the epitopes onE^(ms). This region contains a cysteine on position 422 which might beinvolved in the conformational structure of E^(ms).

To investigate the role of this region, plasmid pPRKc51 harbouring thesmall deletion of amino acids 422-436 was constructed. As is shown inTable 1, immunoperoxidase staining of the transfected cells showed onlyMab binding to E2, and not to E^(ms). These results showed theimportance of this region for the conformation of these E^(ms) epitopes.

Establishment of an SK6 Cell Line Expressing CSFV E^(ms)

SK6 cells were transfected with plasmid pPRKc26 harbouring the CSFVstrain C E^(ms) gene and the histidinol (hisD) resistance gene. After 3weeks, colonies surviving 5 mM histidinol selection were screened forthe expression of E^(ms) by immunostaining the cells with Mab C5specific for CSFV E^(ms). Positive cells were cloned once again toensure clonality.

One of the clones obtained showed expression of E^(ms) in more than 95%of the cells and this clone was named SK6c26. This cell line producedsubstantially higher amounts of E^(ms) than the five other clonesobtained, as was determined by immunostaining. Continuous passaging ofthis cell line SK6c26 in the presence of 5 mM histidinol retainedpersistent expression in more than 95% of the cells for at least 10months (46 passages). Passaging in the absence of histidinol for 10passages resulted in a slight decline of E^(ms)-expressing cells toapproximately 80%.

The stable cell line was further characterized with respect to thebiochemical characteristics of produced E^(ms). The SK6c26 cell linereacted in an IPMA with all tested E^(ms) antibodies (Table 2A). Theamount of E^(ms) in the cell lysates of SK6c26 and SK6 cells infectedwith Flc2 was determined by an indirect ELISA and extrapolated from astandard curve prepared from an immuno-affinity purified preparation ofE^(ms), prepared in insect cells.

Lysates of SK6c26 cells reacted with the E^(ms) specific Mab andpolyclonal antibodies in an indirect ELISA like the wild-type E^(ms)(Table 2B). The amount of E^(ms) produced in the SK6c26 cells (10 ng percm²) was 3-fold lower than SK6 cells infected with Flc2 (30 ng per cm²).The SK6c26 cells and the Flc2-infected SK6 cells possessed comparableRNase activity as measured by an antigen capture RNase assay (Table 2B).The E^(ms) protein of the stable cell line had a similar mobility as thewild-type E^(ms) as determined by SDS-PAGE and was efficiently dimerizedlike the E^(ms) found in virions (Thiel et al., 1991, J. Virol.65:4705-4712) (Table 2B).

Construction and Recovery of C Strain CSFV E^(ms) Recombinant VirusesFlc22 and Flc23

Two recombinants of the E^(ms) gene were replaced in pPRKflc2, thefull-length infectious copy of the CSFV strain C (Moormann et al., 1996,J. Virol. 70: 763-770). Full-length clone pPRKflc22 is derived frompPRKc50 which possessed a deletion of amino acids 422 to 488 in E^(ms)(FIG. 1). The full-length clone pPRKflc23 is derived from pPRKc48 andharbors the five utmost N-terminal amino acids and the six utmostC-terminal amino acids of E^(ms) (deletion of amino acids 273-488) (FIG.1). FIGS. 2A, 2B show a schematic representation of the construction ofthese recombinant E^(ms) full-length clones.

RNA transcribed from the linearized full-length cDNA was transfectedinto the SK6 cell line expressing E^(ms). Four days after transfection,immunoperoxidase staining of the monolayer with Mab b3 did not show E2expression, even with RNA transcribed from pPRKflc2. The amount of E2protein of the recombinant virus might be too low to detect byimmunostaining. Therefore, the cells were passaged to obtain highertitres of viruses. One passage after transfection, wild-type virus Flc2was obtained, while four passages after transfection, expression of E2could be detected with the recombinant clones pPRKflc22 and pPRKflc23.Three to five additional passages were required to obtain a virus titreof approximately 5.5 TCID50/ml and this stock was used for furthercharacterization of the viruses, which were named Flc22 and Flc23 forthe clones pPRKflc22 and pPRKflc23, respectively.

Supernatants from SK6c26 cells infected with Flc22 and Flc23 were usedfor infection of SK6c26 and SK6 cells. Four days after infection, forboth viruses, approximately 50-70% of the SK6c26 cells were positive byE2 immunoperoxidase staining, whereas infection on SK6 cells resulted inonly single cells or pairs of single cells expressing E2. Taking intoaccount that cells infected with CSFV divide normally (once in 24 h),the number of positive cells observed on SK6c26 cells indicatedreplication and secondary spread of the mutated virus. Since only singlecells or pairs of single cells expressed E2 in the SK6 cells, thisindicates that supernatants derived from the SK6c26 cells containinfectious virus which can infect and replicate in SK6 cells but thatthere is no cell-to-cell spread or secondary infection of the mutatedviruses in these cells.

Supernatant or cell lysates of SK6 cells infected with Flc22 and Flc23were used for infection of new SK6 and SK6c26 cells, but this also didnot lead to infected cells. For the infection of pestiviruses, theinteraction of the viral envelope proteins E2 and E^(ms) with thecellular surface are considered to be essential. Due to the absence ofE^(ms) in the SK6 cells, no infectious particles could be formed fromthe viruses Flc22 and Flc23.

Thus, the supernatants of the infected SK6 cells did not containinfectious virus (FIG. 3), whereas supernatant of SK6c26-infected cellswith Flc22 and Flc23 can infect SK6 and SK6c26 cells and, thus,contained infectious virus.

SK6 cells infected with Flc22 and Flc23 derived from the supernatants ofthe SK6c26 cells could be immunostained with Mabs directed against E2,but no positive cells were found with antibodies against E^(ms) (Mabs C5and R716). As control, infection of SK6 cells with supernatant of SK6c26cells infected with Flc2 resulted in a positive immunoperoxidasestaining for both E2 and E^(ms) and secondary infection (FIG. 3).

Transfection of linearized full-length cDNAs pPRKflc22 and pPRKflc23into an SK6-cell line constitutively expressing the bacteriophage T7 RNApolymerase in the cytoplasm of the cell (Van Gennip, 1999, J. Virol.Methods, accepted for publication), resulted in transient expression ofE2 after transfection, but passaging the transfected cells six times didnot result in the recovery of infectious recombinant viruses (data notshown).

Taken together, these results show that the recombinant E^(ms) mutantviruses Flc22 and Flc23 require complementation of E^(ms) by thecomplementing cell line for packaging of the recombinant virus genome toyield infectious virus.

Characterization of Recombinant CSFV Viruses Flc22 and Flc23

To confirm the presence of the mutations in the genomes of Flc22 andFlc23, cellular RNA from infected SK6c26 cells was analyzed with RT-PCRwith CSFV-specific primers. The fragments, after RT-PCR with primersflanking the E^(ms) gene, were of the expected sizes of approximately857, 401, and 1055, respectively for Flc22, Flc23, and Flc2, whereas theRT-PCR product of the E2 gene was for all viruses of the expected sizeof 974 bp. (FIG. 4). The amplification products of the E^(ms) gene weresequenced, and the obtained sequences were as expected. No reversion tothe wild-type or recombination with the E^(ms) gene encoded by the cellline was observed.

Growth kinetics of Flc22 and Flc23 and the wild-type Flc2 weredetermined on the complementing cell line SK6c26. As shown in FIG. 5,the multi-step growth curve of the recombinant viruses Flc22 and Flc23were very similar, but showed a slower growth compared to the parentvirus Flc2. Titres between 5.0-5.8 TCID50/ml were reached by therecombinant viruses after 6 days, whereas the parent strain Flc2 reachedthis titre already within 3 days. The observed titre obtained for Flc2 4days after infection in the complementing cell line is ten-fold lowerthan obtained on the parental cell line SK6 (6.8 TCID50/ml).

Experiments were conducted to determine whether E^(ms) was incorporatedinto the viral envelope. Therefore, virus stocks of Flc2, Flc22 andFlc23 were titrated in the presence of CSFV neutralizing antibodies.Table 3 shows the reduction of virus titre by incubation withneutralizing antibodies. All recombinant viruses were neutralized to thesame extent as the parent virus Flc2 with both the E^(ms)-specific andE2-specific neutralizing polyclonal antibodies. E^(ms) on the viralenvelope of Flc22 could be derived from the complementing cell line aswell as from the recombinant E^(ms) protein encoded by the viral genome,but the recombinant E^(ms) of Flc22 is not recognized by the polyclonalserum R716 (FIG. 3) used for neutralizing the viruses. Thus, the similarneutralization index obtained with this polyclonal serum shows that theamount of E^(ms) derived from the complementing cell line in the viralenvelopes of Flc22 was comparable with those of Flc2 and Flc23.

FIG. 6 shows the amino acid sequence of Flc22 and Flc23 in comparisonwith Flc2. Flc23 is an E^(ms) deletion mutant in which the cleavage sitebetween C-E^(ms) as well as E^(ms)-E1 were left intact; the presence ofthese cleavage sites might influence viability of this virus.

Construction of pPRKflc30, pPRKflc31, pPRKflc32, and pPRKflc33

The results of Table 1 show an important role for the amino acid region422-436 for the conformation of the epitopes of our E^(ms) antibodies.This region contains on position 422 a cysteine, which might beresponsible for epitope recognition. To study the role of this region,we constructed a set of recombinant E^(ms) full-length clones: onelacking amino acids 422-436 (pPRKflc30), a cysteine-to-serine mutant onposition 422 (pPRKflc31), and two additional cysteine-to-serine mutantson positions 405 and 381 (pPRKflc32 and pPRKflc33, respectively) (FIG.1).

RNA transcribed from the linearized full-length cDNAs was transfectedinto the SK6c26 cell line. The cells became positive afterimmunostaining with Mab b3 after passaging the transfected cells. Then,the cells were passaged five times to obtain higher titres of viruses.

Characterization of Recombinant CSFV Viruses Flc30, Flc31, Flc32 andFlc33

Supernatants from SK6c26 cells infected with Flc30, Flc31, Flc32 andFlc33 were used for infection of SK6c26 and SK6 cells. For all virusesapproximately 30-50% of the SK6c26 cells were positive by E2immunoperoxidase staining, four days after infection. For Flc30, Flc31,and Flc33, infection on SK6 cells resulted in only single cells or pairsof single cells expressing E2 and no expression of E^(ms) (FIG. 3). Thisindicates that infection and replication occurred in the SK6 cells, butthat there is no cell-to-cell spread or secondary infection of themutant viruses.

Supernatant of SK6 cells infected with Flc30, Flc31, or Flc33 was usedfor infection of new SK6 and SK6c26 cells, but this did not result ininfected cells. Thus, the supernatants of these infected SK6 cells didnot contain infectious virus (FIG. 3). For the infection ofpestiviruses, the interaction of the viral envelope proteins E2 andE^(ms) with the cellular surface is considered to be essential. Due tothe absence of E^(ms) in the SK6 cells, no infectious particles could beformed from the viruses Flc30, Flc31, and Flc33.

In contrast, infection with Flc32 derived from supernatants of infectedSK6c26 cells showed that 30%-50% of the SK6 cells were infected. Both E2and E^(ms) could be detected by the E2 and E^(ms) antibodies (FIG. 3).Supernatant of SK6 cells infected with Flc32 can infect new SK6 andSK6c26 cells and thus contains infectious virus (FIG. 3).

This indicated that virus Flc32 is capable to replicate in and to infectSK6 cells. Thus, the recombinant virus with a 405 CYS-SER (Flc32)substitution yields infectious virus, whereas the recombinant viruseswith CYS-SER substitutions on position 422 (Flc31) and 381 (Flc33), anda deletion of amino acids 422-436 (Flc30) did not.

These results show an important role for the cysteines at position 422and 381 for functional activity of E^(ms) as cell-to-cell spread,infectivity, and epitope recognition.

pPRKflc23 for the Expression of Heterologous Proteins

pPRKflc23 can be used as vector to incorporate heterologous proteins orfragments of proteins, since the cleavage sites between C-E^(ms) as wellas between E^(ms)-E1 were left intact. FIG. 7 shows a schematicrepresentation of pPRKflc23 harbouring the HA epitope, a non-pestivirussequence. The HA epitope is flanked by the 5 utmost N-terminal aminoacids and 6 utmost C-terminal amino acids of E^(ms).

Example 2

Construction and Characterisation of Recombinant CSFV Strains Flc4 andFlc47

Materials and Methods

Cells & Viruses

Swine kidney cells (SK6-M, EP 0 351 901 B1) were grown in Eagle's basalmedium containing 5% fetal bovine serum, glutamine (0.3 mg/ml), and theantibiotics penicillin (200 U/ml), streptomycin (0.2 mg/ml), andmycostatin (100 U/ml). Fetal bovine serum was tested for the absence ofBVDV and BDV antibodies as described previously (Moormann et al., 1990,Virology 177:184-198).

Recombinant CSFV strain C viruses Flc4 and Flc47 were grown and preparedas described earlier (Moormann et al., 1996, J. Virol. 70: 763-770) witha slight modification; the growth medium of SK6 cells was changed insupplemented Eagle's basal medium. Virus stocks were prepared bypassaging the virus five to ten times on SK6b2 cells. The obtained virustitres ranged from 3.54.5 TCID50/ml.

Construction of a Stable SK6 Cell Line Expressing E2

Plasmid pPRb2 contains the E2 gene of CSFV strain Brescia under controlof the transcriptional and translation signals of expression vectorpEVhisD12 (van Rijn et al., 1992, Vet. Microbiol. 33: 221-230). PlasmidpEVhisD12 is a vector that contains promoter/enhancer sequences of theimmediate early gene of the human cytomegalovirus followed by atranslation initiation codon and the histidinol dehydrogenase gene(hisD) under control of the SV40 early promoter, which can be used as aselective marker Peeters et al., 1992, J. Virol. 66: 894-905).

For transfection of SK6 cells with pPRb2, lipofectamine (20 μg)(Gibco-BRL) was diluted in 50 μl of Optimem-I (Gibco-BRL) mixed withplasmid DNA (1 μg) diluted in 50 μl Optimem-I (Gibco-BRL) and thismixture was allowed to settle for 15 min at room temperature. SK6 cellsgrown in 10 cm² tissue culture plates were washed with Optimem-1. FreshOptimem-I was added (0.5 ml), followed by the DNA transfection mixture.After 20 h of incubation at 37° C., transfected cells were trypsinizedand replated in a ten-fold dilution in microtiter plates in mediumcontaining 10 mM histidinol. Medium was replaced every 3-4 days untilsingle colonies were visible. Wells with individual colonies wererecloned by limited dilution. Expression of E2 was determined byimmunostaining of the cells with Mabs b3 and b4 (Wensvoort et al., 1986,Vet. Microbial. 21: 9-20) directed against conserved epitopes of CSFVE2.

Characterization of the Stable Cell Line SK6b2

E2 expression of the cell line SK6b2 line was tested in animmunoperoxidase staining with E2-specific monoclonal antibodies (Mabs)b3 and b4, (Wensvoort et al., 1986, Vet. Microbiol. 21: 9-20) directedagainst conserved domain A of CSFV E2 and Mabs b6 and b8 (Wensvoort etal., 1986, Vet. Microbiol. 21: 9-20) directed against domain B and C ofBrescia E2.

The amount of E2 was determined by a Ceditest ELISA based on Mab b3 ascoating antibody and horseradish peroxidase conjugated Mab b8 asdetection antibody as described by Colijn et al., (Vet. Microbiol. 59:15-25, 1997).

Construction of Full-Length CSFV Constructs Harbouring E2 DeletionMutants

Plasmid pPRKflc4 is a full-length plasmid harbouring a deletion ofdomain B/C in CSFV-E2 from amino acids 693-746 (FIG. 8A). Therefore, theE2 gene harbouring the deletion from plasmid pPAb16 (van Rijn et al.,1996, J. Gen. Virol. 77: 2737-2745) was inserted in pPRc129 (Moormann etal., 1996, J. Virol. 70: 763-770) by NgoMI/BglII ligation, resulting inplasmid pPRc144. The ClaI/NcoI fragment of pPRc144 was isolated andligated into the ClaI/NcoI digested vector pPRKflc2, formally namedpPRKflc133 (Moormann et al., 1996, J. Virol. 70: 763-770), whichresulted in a full-length clone named pPRKflc4.

Plasmid pPRKflc47 is a full-length plasmid harbouring a deletion of thecomplete CSFV-E2 gene from amino acids 689-1062 (FIG. 8B). Therefore, aPCR fragment harbouring the deletion was amplified from plasmid pPRKflc2with forward primer p1195 (5′-GGC TGT TAC TAG TAA CTG GGG CAC AAG GCTTAC CAT TGG GCC AGG GTG-3′ (SEQ ID NO:20)) at position 2412 of thenucleotide sequence of the C-strain and reverse primer p403 (5′-CCC GGGATC CTC CTC CAG TTT TTT GTA AGT GGA-3′ (SEQ ID NO:21)) at nucleotideposition 5560. The SpeI/AflII fragment was isolated and ligated inSpeI/AflII-digested pPRc144, resulting in plasmid pPRKc58. The ClaI/NcoIfragment of pPRKc58 was isolated and ligated into the ClaI/NcoI digestedvector pPRKflc2, which resulted in full-length clone named pPRKflc47.

Isolation of Recombinant Viruses Flc4 and Flc47

Plasmid DNA from pPRKflc4 and pPRKflc47 was purified on columns (Qiagen)and linearized with XbaI. The DNA was extracted with phenol-chloroform,precipitated with ethanol, and dissolved in water. RNA was transcribedfrom the linearized plasmid (1 μg) in a 100 μl reaction volumecontaining 40 mM TrisHCl (pH 7.5), 6 mM MgCl2, 2 mM spermidine, 10 mMdithiothreitol, 40 U rRNAsin (Promega), 0.5 mM each rNTP, and 35 U T7RNA polymerase (Pharmacia). After 1 h incubation at 37° C., 10 URNase-free DNaseI (Pharmacia) was added and the mixture was incubatedfor another 15 min. RNA was extracted with phenol-chloroform,precipitated with ethanol, and dissolved in 10 μl water.

For RNA transfection, 10 μg Lipofectin was diluted in 50 μl ofOptimem-I. After a 45 min. incubation at room temperature, 1 μg RNAdiluted in 50 μl Optimem-I was added and incubated for an additional 15min. SK6b2 cells grown in 10 cm² tissue culture plates were washed withOptimem-I and incubated with the RNA transfection mixture for 4 h at 37°C. Then, the wells were supplied with fresh medium and incubated for 4days at 37° C. RNA transfection was performed in duplicate. One samplewas immunostained with Mab C5 (Wensvoort 1998, Thesis, University ofUtrecht) specific for C strain E^(ms). When the E^(ms) immunostainingproved to be positive, the duplicate sample was passaged and split intotwo samples. One of these samples was used for immunostaining four daysafter passaging. From wells in which E^(ms) expression was observed,supernatant was applied onto fresh SK6 cells to determine the presenceof infectious virus. After four days, the monolayers were fixed andimmunostained as described above.

Characterization of Flc4 and Flc47

Viruses were characterized by an immunoperoxidase monolayer assay. Forthis, SK6 cells were infected with viruses Flc2, Flc4 and Flc47. Afterincubation for 4 days at 37° C., monolayers were immunostained with Mabsspecific for CSFV E2 (Mabs b3/b4) and CSFV E^(ms) Mab C5.

Results

Establishment of an SK6 Cell Line Expressing CSFV E2

SK6 cells were transfected with plasmid pPRb2 containing the CSFV strainBrescia E2 gene and the histidinol (hisD) resistance gene. After 2weeks, colonies surviving 10 mM histidinol selection were recloned bylimited dilution and screened for the expression of E2 by immunostainingthe cells with Mab b3 specific for CSFV E2. This clone was named SK6b2.The biochemical properties of the produced E2 of the SK6b2 cell linewere characterised. SK6b2 cells reacted with the E2-specific Mabs in animmunoperoxidase assay (Table 4A) and in an indirect ELISA likewild-type E2 (Table 4B). The amount of E2 in the cell lysates of SK6b2and SK6 cells infected with Flc2 was determined by an indirect ELISA andextrapolated from a standard curve prepared from an immuno-affinitypurified preparation of E2 prepared in insect cells. The amount of E2determined in the SK6b2 cell line (115 ng per cm²) was 3-fold higherthan that of SK6 cells infected with Flc2 (30 ng per cm²). The E2protein of the stable cell line had a similar mobility as the wild-typeE2 as determined by SDS-PAGE and was efficiently dimerized like the E2found in virions (Thiel et al., 1991, J. Virol. 65:4705-4712) (Table4B).

Construction and Recovery of C Strain CSFV E2 Recombinant Viruses

Previous studies have shown that E2 consists of the two antigenic unitsA and B/C and that the separate antigenic units of E2 can protect pigsagainst classical swine fever (van Rijn et al., 1996, J. Gen. Virol. 77:2737-2745). Therefore, two deletion mutants were constructed: plasmidpPRKflc4, which possesses a deletion in E2 of domain B/C between aminoacids 693-746 and pPRKflc47, in which the entire E2 gene between aminoacids 689-1062 was deleted. FIGS. 8A, 8B show a schematic representationof the construction of these recombinant E2 full-length clones.

RNA transcribed from the linearized full-length cDNA, was transfectedinto the SK6b2 cell line expressing E2. The cells were positive afterimmunostaining with Mab C5. Then, the transfected cells were passaged toobtain higher titres of viruses. Supernatants isolated from serialpassages contained infectious viruses. Between five to ten passages wererequired to obtain a virus titre of approximately 3.54.5 TCID50/ml andthis stock was used for further characterisation of the viruses.

Characterisation of Recombinant E2 CSFV Viruses

Supernatants from the SK6b2 cells were used for infection of SK6 andSK6b2 cells to characterize Flc4 and Flc47. Four days after infection,approximately 30-50% of the SK6b2 cells were positive by E^(ms)immunoperoxidase staining.

Infection on SK6 cells resulted in only single cells or pairs of singlecells expressing E^(ms) four days after infection. This indicates thatinfection and replication occurred in the SK6 cells, but that there isno cell-to-cell spread or secondary infection of the recombinantviruses.

Supernatant of the Flc4- and Flc47-infected SK6 cells were used forinfection of new SK6 and SK6b2 cells, but this did not result intoinfected cells. Thus, the supernatants of these infected SK6 cells donot contain infectious virus (Table 5). For the infection ofpestiviruses, the interaction of the viral envelope proteins E2 andE^(ms) with the cell surface are considered to be essential. Due to theabsence of E2 in the SK6 cells, no infectious particles could be formed.Infection of SK6 cells with Flc2 from infected SK6b2 cells resulted in apositive immunoperoxidase staining for E2 and E^(ms) and in secondaryinfection of the virus.

SK6 cells infected with the viruses Flc4 and Flc47 could beimmunostained with Mab C5 directed against E^(ms), whereas only Flc4 andFlc2 reacted with Mab b3, directed against the A domain of CSFV E2(Table 5). As expected, Flc47 was completely negative for the Mabsagainst the A, B and C domains (Table 5).

Transfection of in vitro RNA derived from pPRKflc4 and pPRKflc47 intothe SK6-cell line resulted in transient expression of E^(ms), butpassaging the transfected cells six times did not result in the recoveryof recombinant viruses. This indicated that the recombinant E2 virusesFlc4 and Flc47 require complementation of E2 to obtain infectious virus.

Example 3 Animal Experiment: 298-47042-00/98-06

Pigs Vaccinated with Flc22 and Flc23 are Protected Against a LethalChallenge with Virulent CSFV Strain Brescia

Materials and Methods

Animals

Pigs of 6-7 weeks of age from conventional sows were obtained. Pigs wererandomly divided in groups and housed in separate stables of the highlycontainment facilities of ID-DLO. The animals were fed once a day, in atrough, with complete food pellets, and could drink water from a nipplead libitum.

Vaccination and Challenge

The pigs were divided into two groups of 2 pigs. The pigs in group Awere vaccinated with strain Flc23; the pigs in group B were vaccinatedwith strain Flc22. The pigs were vaccinated via several routes ofinoculation. The pigs were sedated and placed on their backs beforeinoculation with a virus suspension (2 ml containing 105 TCID50/ml)which was instilled dropwise into the nostrils. In addition, twomilliliters of virus suspension was inoculated intravenously, 2 ml ofvirus was inoculated intradermally. The vaccines were emulsified in awater-oil-water adjuvant (Hulst et al., 1993, J. Virol. 67:5435-5442),and 2 ml of this vaccine was inoculated intramuscularly in the neckbehind the ears. In total, each pig received 8 ml of vaccine,corresponding with 8×10⁵ TCID₅₀/ml.

The pigs were challenged intranasally with 100 50% lethal doses (=100LD₅₀) of CSFV strain Brescia 456610 (Terpstra and Wensvoort, 1988, Vet.Microbiol. 16:123-128) four weeks after vaccination. Viral contents ofthe vaccine inoculum were determined by titration of a sample takenafter return from the stable. The pigs were euthanized 7 weeks afterchallenge.

Clinical Observation

The pigs were checked daily by the animal technicians, abnormal findingswere recorded and if necessary the supervising veterinarian was called.Each group was observed at least 15 minutes per day before and duringfeeding and cleansing of the stable. A reduction in food uptake of thegroup or an individual animal was noted. Body temperatures were recordedduring several days before and up to 20 days after challenge.

Blood Analysis After Challenge

EDTA-blood samples were collected on days 1, 2, 6, 9, 12 and 15 afterchallenge to monitor changes of leukocyte and thrombocyte numbers in theblood. A decrease in the number of leukocytes (leucopenia) andthrombocytes (thrombocytopenia) is one of the typical signs of CSF.Normal cell counts for white blood cells and thrombocytes inconventional swine range between 11-23 109/l and 320-720 109/l,respectively. Both ranges mentioned vary in each pig. The blood cellanalyses were performed with a Medonic CA 570 coulter counter.Leucopenia and thrombocytopenia were defined as cell/platelets countsconsiderably lower than the minimum number mentioned above, preferablyfor more than one day.

Virus Isolation and Viral Antigen Detection

Virus isolation: Peripheral blood leukocytes were extracted fromEDTA-blood samples taken on day —1, 2, 6, 9, 12 and 15 after challengeto monitor viraemia. The samples were stored at −70° C. The presence ofCSFV in the leukocytes was examined as follows. In an M24 plate(Greiner), 300 μl (containing approximately 5×106 cells) of a swinekidney cell (SK6) suspension was added to each well and cultured at 37°C. and 5% CO₂ in a humid chamber for 24 h. After 24 h, the medium wasremoved and 300 μl of an undiluted freeze/thawed leukocyte sample wasadded per well. After one hour of incubation at 37° C. and 5% CO₂, thesample was removed. The monolayer was then washed by adding and removing400 μl of culture medium (Eagle basal medium). Subsequently, 800 μl ofculture medium (Eagle basal medium with 5% fetal bovine serum (FBS),free of pestivirus antibodies, and 1% of an antibiotic stock containingglutamine (0.3 mg ml⁻¹), penicillin (200 units ml⁻¹), streptomycin (0.2mg ml⁻¹) and mycostatin (100 U ml⁻¹)) was added per well. After fourdays, the monolayers were washed in 10% NaCl solution, dried for 1 h at800 C., incubated with a buffered solution containing CSFV-specificconjugated antibodies, washed and stained. The monolayers were readmicroscopically for stained cells. Results were expressed as positive ornegative for virus.

IFT: At post-mortem, tissue samples were collected from tonsil, spleen,kidney, and ileum, and were tested by direct immunofluorescent technique(Ressang and De Boer, 1967, Tijdschrift voor Diergeneeskunde 92:567-586)for the presence of viral antigen. Cryostat sections (4 μm thick, twoper organ) from these tissue samples were fixed and incubated with apolyclonal swine anti-pestivirus FITC-conjugated serum. After washing,the sections were read under a fluorescence microscope. Results wereexpressed as positive (=fluorescence) or negative (=no fluorescence).

Serological Response

Serum blood samples of all pigs except the controls were collected atone week intervals after challenge during 6 weeks. Samples were storedat −20° C. and assayed in a CSFV-specific (Terpstra and Wensvoort, 1984,Vet. Microbiol. 33:113-120) virus neutralisation test (NPLA), in theCeditest ELISA for detecting CSFV-specific antibodies against E2 (Colijnet al., 1997, Vet. Microbiol. 59:15-25), and in a Ceditest ELISA for thedetection of antibodies against E^(ms) (de Smit et al., in prep).

CSFV-specific neutralising antibody titres in serum were determined in amicrotiter system. Serial two-fold dilutions of serum were mixed with anequal volume of a CSFV (strain Brescia) suspension which contained30-300 TCID50. After incubation for 1 hour at 37° C. in a CO₂ incubatorapproximately 25,000 PK-15 cells per well were added. After four days,the microtiter plates were treated as mentioned above and readmicroscopically. The CSFV neutralising titre was expressed as thereciprocal of the highest dilution that neutralised all viruses.

The CSFV E2-ELISA was performed according to the instructions of themanufacturer (Colijn et al., 1997, ibid.). The CSFV E^(ms)-ELISA wasperformed as follows. Test sera (30 μl) are pre-incubated with CSFVE^(ms)-antigen (70 μl of a working dilution of baculovirus expressedE^(ms) of CSFV strain Brescia) in a 96-well non-coated microtiter platecontaining 45 μl of ELISA buffer for 30 min at 37° C. Thereafter, 50 μlof this pre-incubation mix is added to a microtiter plate coated withthe E^(ms)-specific monoclonal antibody 137.5, and containing 50 μl of aworking solution of the horseradish peroxidase conjugatedE^(RNS)-specific monoclonal antibody 140.1.1. The plates are incubatedfor 1 h at 37° C., washed six times with 200 μl of washing solution, andincubated for 30 min at room temperature with 100 μl of a ready-to-usechromogen(3,3′,5,5′-tetramethylbenzidine)/substrate solution. The colorreaction is stopped by adding 100 μl of a 0.5 M H2SO4 solution, and theoptical density was measured at 450 nm using an Easy Readerspectrophotometer (SLT Vienna).

Results

Clinical Observation, Viral Antigen Detection, Leukocyte/ThrombocyteCounts

After vaccination, none of the animals developed clinical signs or fever(Table 6). Both group A and B pigs developed a mild fever (40° C.<T<41°C.) for three days, starting 3 days after challenge. None of thevaccinated pigs, either in group A (Flc23) or B (Flc22) developedleucopenia or thrombocytopenia (Table 6), although six days afterchallenge a slight drop in the thrombocyte count and leukocyte count wasobserved for most of the pigs. In both groups A and B, no virus wasdetected in the leukocytes. Moreover, the organs of all pigs wereIFT-negative at the end of the experiment, indicating the absence ofpersistent infections.

Serological Response

After vaccination of the pigs in group A (Flc23) and group B (Flc22), noCSFV-specific antibodies were detected with the E2-ELISA (Table 7) andthe E^(ms) ELISA (Table 8). This finding was consistent with the NPLAresults: all vaccinated pigs remained negative for neutralisingantibodies against CSFV (Table 9).

After challenge inoculation, maximum inhibition percentages wereobserved in the E2-ELISA in all inoculated pigs. Also, all four pigsseroconverted in the E^(ms) ELISA. In the neutralising antibody assays,all inoculated pigs showed high titres against CSFV. These resultsclearly show that both Flc22 and Flc23 vaccines protect against a lethalchallenge of virulent Brescia, and can be discriminated from infectedanimals by CSFV-specific E^(ms)-ELISA.

TABLE 1 IPMA on SK6 monolayers transfected with E^(ms) expressionplasmids harbouring the nucleotide sequence of the autoprotease andstructural genes (Npro —C-recombinant E^(ms)-E1-E2) E^(ms) E2 PlasmidDeletion* C5 140.1 R 716 137.5 b3 b4 pPRK83 None + + + + + + pPRKc49 436to 488 + + + + + + pPRKc50 422 to 488 − − − − + + pPRKc51 422 to 436 − −− − + + PPRKc48 272 to 488 − − − − + + *amino acid numbering of CSFVstrain C (Moormann et al., 1996, J. Virol. 70: 763-770)

TABLE 2A IPMA reactivity of the SK6c26 and SK6 cells with CSFV E^(ms)antibodies CSFV antibodies Cells Mab C5 Mab 140.1 Mab 137.5 R 716SK6c26 + + + + SK6 − − − −

TABLE 2B Comparison of E^(ms) of SK6c26 and SK6 infected cells with Flc2RNase activity Cells ng E^(ms).cm^(−2a) (A₂₆₀.min⁻¹.mg⁻¹)^(b)Dimerization^(c) SK6c26 10 130 + SK6 <0.1 0 − SK6 Flc2 infected 30 171 +^(a)The amount of E^(ms) in the cell lysates per cm² was extrapolatedfrom a standard curve prepared from an immuno-affinity purifiedpreparation of E^(ms) produced in insect cells. ^(b)The RNase activitywas determined as A₂₆₀ units per mg E^(ms) per min as described by Hulstet al., 1998, J. Virol. 72:151-157. ^(c)detection of dimers of E^(ms) bynonreducing SDS-PAGE

TABLE 3 Neutralization of CSF viruses by antibodies Virus neutralizingreduction (log TCID₅₀/ml) with serum^(a) Virus 716 (directed againstE^(ms))^(b) 539 (directed against E2)^(c) Flc2 −3.0 −1.75 Flc22 −3.0−1.75 Flc23 −3.25 −1.0 ^(a)Log TCID₅₀/ml reduction of CSFV titers due tothe presence of serum ^(b)rabbit serum prepared against E^(ms) of CSFVstrain C ^(c)pig serum specifically directed against E2 of CSFV strainBrescia

TABLE 4A Reactivity of SK6b2 and SK6 cells with CSFV E2 Mabs Reactivityof CSFV antibodies in IPMA Cells b3 b8 b6 SK6b2 + + + SK6 − − −

TABLE 4B Characterization of the SK6b2 cell line Cells Dimerization^(a)ng E2.cm^(−2b) SK6b2 + 115 SK6 − 0 SK6Flc2 infected + 38^(a)Dimerization of E2 was determined on SDS-PAGE under nonreducingconditions ^(b)The amount of E2 in the cell lysates per cm² as measuredin the E2 ELISA was extrapolated from a standard curve prepared from animmuno-affinity purified preparation of E2 produced in insect cells.

TABLE 5 Characterization of recombinant E2 viruses on SK6 cells IPMAreactivity on Infectious virus SK6 cells recovered^(a) SK6b2 SK6 VirusC5 b3 b6 cells cells Flc2 + + + + + Flc4 (deletion B/C domains of + +− + − E2) Flc47 (deletion E2) + − − + − ^(a)Infectious virus isrecovered if supernatants of infected cells can infect new SK6 and SK6b2cells. Spread of virus via cell-to-cell spread and spread of virus dueto division of cells is not considered due to infection of virus.

TABLE 6 Results of virus isolation, cytopenia and fever after challengewith CSFV pig # days Group no. with fever^(a) Viremia IFT Cytopenia^(b)death A 469 3 − − − − Flc23 476 5 − − − − B 477 5 − − − − Flc22 478 6 −− − − ^(a)Fever: body temperature >40° C. ^(b)Cytopenia:thrombocytopenia and/or leucopenia

TABLE 7 Results of the Ceditest ® ELISA for the detection of CSFV-E2antibodies^(a) days post challenge A 469 0 0 27 34 17 61 100 100 100 4760 0 0 0 18 97 100 100 100 B 477 0 0 11 17 32 93 100 100 100 478 0 0 1524 34 51 100 100 100 ^(a)The Ceditest ® E2-ELISA specifically detectsantibodies against envelope protein E2 of CSFV. Test results areexpressed as the percentage inhibition of a standard signal; <30% isnegative , 30-50% inhibition is doubtful, >50% inhibition is positive.

TABLE 8 Results of the Ceditest ® ELISA for the detection of CSFV-E^(ms)antibodies^(a) Animal days post challenge Group no. −28 −15 −1 6 12 1927 33 49 A. Flc23 469 17 29 26 22 76 80 81 74 83 476 15 28 25 14 70 7681 69 79 B Flc22 477 13 30 13 20 70 54 49 50 62 478 12 34 35 22 68 84 8187 92 ^(a)The Ceditest ® E^(ms)-ELISA specifically detects antibodiesagainst envelope protein E^(ms) of CSFV. The test results are expressedas the percentage inhibition of a standard signal; <50% is negative,=50% is positive

TABLE 9 Results of the NPLA for the detection of CSFV Brescia-specificneutralizing antibodies Animal Days post challenge Group No. −28 −21 −15−9 −1 6 12 19 27 33 49 A 469 <10 <10 <10 <10 1580 >1280 >1280 >1280 >1280 7680 Flc23 476 <10 <10 <10 <10 <1080 >1280 >1280 >1280 >1280 7680 B 477 <10 <10 <10 <10 1080 >1280 >1280 >1280 >1280 >20480 Flc22 478 <10 <10 <10 <10 <10480 >1280 >1280 >1280 >1280 7680

1. A recombinant nucleic acid that, upon inoculation of an animal, canelicit an immune response directed against immunodominant parts ofstructural proteins of a pestivirus without having the ability to spreadthrough the inoculated animal, said recombinant nucleic acid comprisinga genome of a pestivirus encoding at least one structural protein or animmunodominant part thereof, said genome having a functional deletion ina sequence encoding at least one protein related to viral spread.
 2. Acell permissive for pestivirus infection comprising: a nucleic acidconstruct encoding at least one pestiviral protein or substantial partthereof related to viral spread; and a pestiviral genome containing adefect complemented by said construct; wherein said cell allows forpackaging of said genome and pestiviral protein or substantial partthereof in a pestivirus-like particle.
 3. The cell of claim 2, whereinsaid at least one pestiviral protein or substantial part thereof relatedto viral spread is stably expressed.
 4. A process of makingpestivirus-like particles comprising: transfecting a cell with arecombinant nucleic acid comprising a pestiviral genome having afunctional deletion in a sequence encoding at least one pestiviralprotein related to viral spread, said genome being capable of RNAreplication in a suitable cell and encoding at least one structuralprotein of pestivirus or at least one immunodominant part thereof,wherein said cell comprises a second nucleic acid construct encoding atleast one pestiviral protein or a substantial part thereof related toviral spread and allowing packaging of said recombinant pestiviralgenome in a pestivirus-like particle; allowing said pestiviral genome toreplicate in said cell; allowing the replicated recombinant pestiviralgenome to package into a pestivirus-like particle comprising the proteinencoded by the second nucleic acid; and harvesting said pestivirus-likeparticles.
 5. The process according to claim 4 wherein the secondnucleic acid construct encoding the at least one pestiviral protein orsubstantial part thereof related to viral spread is stably expressed. 6.A process of making a vaccine comprising: transfecting a cell with arecombinant nucleic acid comprising a pestiviral genome having afunctional deletion in a sequence encoding at least one pestiviralprotein related to viral spread, said genome being capable of RNAreplication in a suitable cell and encoding at least one structuralprotein of pestivirus or at least one immunodominant part thereof;wherein said cell comprises a second nucleic acid construct encoding atleast one pestiviral protein or a substantial part thereof related toviral spread and allowing packaging of said recombinant pestiviralgenome in a pestivirus-like particle; allowing said pestiviral genome toreplicate in said cell; allowing the replicated recombinant pestiviralgenome to package into a pestivirus-like particle comprising the proteinencoded by the second nucleic acid; harvesting said pestivirus-likeparticles; and preparing a suspension of said pestivirus-like particlesin a suitable diluent.
 7. The process according to claim 6 comprisingcombining said suspension with an adjuvant.
 8. The process according toclaim 6, wherein said functional deletion in the sequences encoding atleast one structural protein or pestivirus or at least oneimmunodominant part thereof comprises a functional deletion in E² orE^(ms).